Characterization of EstCOo8 and EstC34, intracellular esterases, from the wine-associated lactic acid bacteria Oenococcus oeni and Lactobacillus hilgardii.

AIM: To clone and characterize two related intracellular esterases from Oenococcus oeni and Lactobacillus hilgardii under wine-like conditions. METHODS AND RESULTS: The published genome sequences for O. oeni and Lact. hilgardii were used to identify, clone and purify putative esterase genes from these species designated EstCOo8 and EstC34, respectively. Both esterases are members of family V of lipolytic enzymes. However, EstC34 contains an SGSLG nucleophilic elbow structural motif instead of the usual GGSLG motif which is conserved in other lactic acid bacteria. Both esterases exhibited greatest specificity for C(2) -C(4) pNP-linked substrates and retained activity under wine-like conditions. EstCOo8 had an optimum temperature, pH, and ethanol concentration of 40°C, 5.5 and 6% (v/v), respectively. Whereas EstC34 had an optimum temperature, pH and ethanol concentration of 50°C, 5.0 and 10% (v/v), respectively. CONCLUSIONS: Both esterases were stable and retained activity under conditions that would be encountered in wine. They have the potential to reduce short-chain ethyl esters such as ethyl acetate. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information that might help improve the performance of LAB during malolactic fermentation in wine in the future, either by strain selection, optimization or direct enzyme addition.

A novel methodology independent of fermentation rate for assessment of the fructophilic character of wine yeast strains.

The yeast Saccharomyces cerevisiae has a fundamental role in fermenting grape juice to wine. During alcoholic fermentation its catabolic activity converts sugars (which in grape juice are a near equal ratio of glucose and fructose) and other grape compounds into ethanol, carbon dioxide and sensorily important metabolites. However, S. cerevisiae typically utilises glucose and fructose with different efficiency: glucose is preferred and is consumed at a higher rate than fructose. This results in an increasing difference between the concentrations of glucose and fructose during fermentation. In this study 20 commercially available strains were investigated to determine their relative abilities to utilise glucose and fructose. Parameters measured included fermentation duration and the kinetics of utilisation of fructose when supplied as sole carbon source or in an equimolar mix with glucose. The data were then analysed using mathematical calculations in an effort to identify fermentation attributes which were indicative of overall fructose utilisation and fermentation performance. Fermentation durations ranged from 74.6 to over 150 h, with clear differences in the degree to which glucose utilisation was preferential. Given this variability we sought to gain a more holistic indication of strain performance that was independent of fermentation rate and therefore utilized the area under the curve (AUC) of fermentation of individual or combined sugars. In this way it was possible to rank the 20 strains for their ability to consume fructose relative to glucose. Moreover, it was shown that fermentations performed in media containing fructose as sole carbon source did not predict the fructophilicity of strains in wine-like conditions (equimolar mixture of glucose and fructose). This work provides important information for programs which seek to generate strains that are faster or more reliable fermenters.

[Selenium deficiency of west Bohemia population]. / Deficit selenu v populaci západních Cech.

To estimate status and intake of selenium in inhabitants of the most Western region of the Czech Republic (Cheb region) 241 serum, 404 urine and 30 hair samples from randomly selected persons in the age between 6 and 65 years is performed. Serum and hair samples were analysed by means of instrumental neutron activation analysis (INAA), while Se in urines was detected by means of fluorimetry. Urine iodine was determined in the same group by Sandell-Kolthoff method for the possibility to detect concomitant Se and I deficiency and/or correlations between these two essential trace elements necessary for metabolism of thyroid hormones. Average values of Se indexes are low (55.4 +/- 13.8 microg Se/L serum; 15.4 +/- 5.7 microg Se/L urine; 13.6 +/- 6.0 microg Se/g creatinine; 0.268 +/- 0.051 microg Se/g hair) and prove Se deficiency in the searched population. Statistical evaluation of Se in subgroups of boys, girls, men and women proved significant differences as far as age is concerned, gender differences were found only between boys and girls. Some significant and highly significant differences were found also in subgroups according age and gender (males and females in the age of 6, 10, 13, 18-35, 36-49 and 50-65 years). On the other hand, urine iodine average value (126 +/- 65 microg/L) is on the lower optimum level. By the use of correlation analysis, slight but significant correlations were found between Se and I in urine and some of thyroid hormone parameters and their influence on the organism.

Filtration, haze and foam characteristics of fermented wort mediated by yeast strain.

AIMS: To investigate the influence of the choice of yeast strain on the haze, shelf life, filterability and foam quality characteristics of fermented products. METHODS AND RESULTS: Twelve strains were used to ferment a chemically defined wort and hopped ale or stout wort. Fermented products were assessed for foam using the Rudin apparatus, and filterability and haze characteristics using the European Brewing Convention methods, to reveal differences in these parameters as a consequence of the choice of yeast strain and growth medium. CONCLUSIONS: Under the conditions used, the choice of strain of Saccharomyces cerevisiae effecting the primary fermentation has an impact on all of the parameters investigated, most notably when the fermentation medium is devoid of macromolecular material. SIGNIFICANCE AND IMPACT OF THE STUDY: The filtration of fermented products has a large cost implication for many brewers and wine makers, and the haze of the resulting filtrate is a key quality criterion. Also of importance to the quality of beer and some wines is the foaming and head retention of these beverages. The foam characteristics, filterability and potential for haze formation in a fermented product have long been known to be dependant on the raw materials used, as well as other production parameters. The choice of Saccharomyces cerevisiae strain used to ferment has itself been shown here to influence these parameters.

Screening of Lactobacillus spp. and Pediococcus spp. for glycosidase activities that are important in oenology.

AIMS: To assess glycosidase activities from a range of Lactobacillus and Pediococcus species and characterize these activities under conditions pertinent to the wine industry. METHODS AND RESULTS: Lactic acid bacteria were cultured in MRS broth supplemented with apple juice before being harvested, washed and assayed for glycosidase activity using p-nitrophenol-linked substrates. All strains exhibited a detectable capacity for the hydrolysis of the beta- and alpha-d-glucopyranosides. The magnitude of these activities and their response to the physico-chemical parameters investigated varied in a strain-dependent manner. The use of an assay buffer with a pH below 4 generally resulted in a reduced hydrolysis of both substrates while temperature optima ranged between 35 and 45 degrees C. The effect of the inclusion of ethanol in the assay buffer (up to 12%, v/v) ranged from near complete inhibition to increases in activity approaching 80%. With the clear exception of a single strain, glucose and fructose (0.1-20 g l(-1)) acted as inhibitors. An assessment of glycosidase activity during simultaneous exposure to glucose and ethanol at a pH of 3.5 suggested that ethanol decreased loss of activity under these wine-like conditions. CONCLUSIONS: Lactobacillus spp. and Pediococcus spp. possess varying degrees of beta- and alpha-d-glucopyranosidase activities, which in turn are influenced differently by exposure to ethanol and/or sugars, temperature and pH. Several strains appeared suited for further evaluation under winemaking conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This work highlights the fact that strains of Lactobacillus and Pediococcus have the potential to influence the glycoside composition of wine. Tailoring of wine may therefore be possible through selective application of strains or enzymatic extracts thereof.

INAA of serum zinc of inhabitants in five regions of the Czech Republic.

To investigate the Zn status of inhabitants of the Czech Republic, 1155 serum and 132 hair samples were analyzed for zinc content. Analyzed material was obtained from randomly selected volunteers of both sexes in the age range 6-65 yr. Subpopulations from five regions were included in the study. Analyses of both materials were performed by Instrumental Neutron Activation Analysis (INAA). Coanalyses of Standard Reference Materials (SRM) for quality control were performed. The results (mean 910 +/- 276 micrograms Zn/L serum and 189 +/- 45 micrograms Zn/g hair) demonstrate satisfactory zinc status of the searched population. Significant interregional differences and age-dependent differences, as well as sex-dependent differences have been detected by the use of correlation analysis. On the basis of these results, serum Zn concentration results of individual categories according to age and sex were evaluated, and on the grounds of mean +/- 2 standard deviations, indicative intervals were calculated. The frequency of individual serum Zn concentrations proved that the population of the Czech Republic does not suffer from severe zinc deficiency. On the other hand, about one-third of our inhabitants has their serum Zn concentrations below the cutoff value of 800 micrograms Zn/L serum, which means a marginal or mild Zn deficit of the organism.

Characterization of selenium status of inhabitants in the region Usti nad Orlici, Czech Republic by INAA of blood serum and hair and fluorimetric analysis of urine.

Selenium is an essential trace element and its insufficient status may cause serious health complications for both individuals and the whole populations. To investigate the selenium status of the subpopulation in northeastern Bohemia represented by the region Ustí nad Orlicí, 253 serum, 469 urine, and 31 hair samples from 470 randomly selected volunteers between 6 and 65 yr of age have been analyzed for selenium concentration. Serum and hair Se were detected by instrumental neutron activation analysis (means: 55 +/- 11 micrograms Se/L sera, 0.268 +/- 0.040 microgram Se/g hair). Urine Se was measured by fluorimetry (12 +/- 5 micrograms Se/L urine) with coanalyses of Lyphocheck urine, SRM Urine 2670, and Seronorm urine for quality control of the method. Results proved significant age-dependent differences, but gender differences were not significant. The frequency plot of serum Se proved maximal frequencies in adults between 55 and 70 micrograms Se/L and in children in the range 45-55 micrograms Se/L. The same plots of urine Se for both age groups showed maximal frequency in the limits 8-15 micrograms Se/L. All indices used (Se in serum, urine, and hair) confirmed mild to severe selenium deficiency in the population of the region.

Determination of sulphite reductase activity and its response to assimilable nitrogen status in a commercial Saccharomyces cerevisiae wine yeast.

The liberation of H2S is a common problem afflicting wine fermentation. Sulphite reductase activity of a commercial wine yeast was investigated to define its involvement in this process. The activity studied here differed from those characterized previously from cider and bakers' yeasts by displaying a greater sensitivity to cold, low ionic strength and possibly, proteolytic action. These differences necessitated the development of a new method of quantification. Through this method, the onset of H2S liberation was shown not to be a result of variations in the levels of sulphite reductase activity. Thus, high levels of activity which occurred during the exponential phase of growth were not necessarily accompanied by the liberation of H2S. Similarly, nitrogen-starved cultures which liberated H2S showed no corresponding increase in sulphite reductase activity from prestarvation levels. In fact, rates of H2S liberation from cultures and in enzyme assays agreed closely. A short-term independence of sulphite reductase activity from culture nitrogen status was therefore evident. The only influence of nitrogen was achieved in its absence when enzyme activity decayed with a half-life (4.25 h) which was comparable to that induced by the presence of cycloheximide (5.75 h). A proposed transcriptional control mechanism mediated by methionine derivatives was only partly effective in this strain although an in vitro inhibitory effect of methionine was implicated. These data therefore support the notion that H2S liberation in response to nitrogen starvation stems from a failure of metabolism to sequester H2S which continues to be formed, at least initially, at prestarvation rates.

Regulation of hydrogen sulfide liberation in wine-producing Saccharomyces cerevisiae strains by assimilable nitrogen.

Saccharomyces cerevisiae wine-producing yeast cultures grown under model winemaking conditions could be induced to liberate hydrogen sulfide (H2S) by starvation for assimilable nitrogen. The amount of H2S produced was dependent on the yeast strain, the sulfur precursor compound, the culture growth rate, and the activity of the sulfite reductase enzyme (EC 1.8.1.2) immediately before nitrogen depletion. Increased H2S formation relative to its utilization by metabolism was not a consequence of a de novo synthesis of sulfite reductase. The greatest amount of H2S was produced when nitrogen became depleted during the exponential phase of growth or during growth on amino acids capable of supporting short doubling times. Both sulfate and sulfite were able to act as substrates for the generation of H2S in the absence of assimilable nitrogen; however, sulfate reduction was tightly regulated, leading to limited H2S liberation, whereas sulfite reduction appeared to be uncontrolled. In addition to ammonium, most amino acids were able to suppress the liberation of excess H2S when added as sole sources of nitrogen, particularly for one of the strains studied. Cysteine was the most notable exception, inducing the liberation of H2S at levels exceeding that of the nitrogen-depleted control. Threonine and proline also proved to be poor substitutes for ammonium. These data suggest that any compound that can efficiently generate sulfide-binding nitrogenous precursors of organic sulfur compounds will prevent the liberation of excess H2S.